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Characterization of phthalate presence in cattle environment and its effects on epigenetic markers expression and steroidogenesis in bovine granulosa cells
AuthorPena Bello, Camilo Andres
AdvisorSchutz, Luis F
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Farm animals are now more likely to be exposed to environmental pollutants like phthalates as result of intensifying livestock production systems. Phthalate exposure may cause impairments in human and animal health and fertility. However, research into the occurrence of phthalates in cow operations is scarce. Also, studies on the impact of Di-isobutyl phthalate (DIBP) on female fertility are limited in phthalate research. The first project aimed to determine the effects of DIBP in bovine granulosa cells (GC) proliferation, steroidogenesis, and epigenetic and inflammation pathways. Using in vitro cell culture of bovine GC, DIBP treatment at levels detected in human blood (0 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL) combined with hormones that regulate ovarian folliculogenesis: Follicle-Stimulating Hormone (FSH) or FSH + Insulin Growth Factor 1 (IGF1) were tested. Estradiol production was assessed by ELISA, cell proliferation was determined by cell counting, and relative mRNA abundance was evaluated by RT-qPCR. Cell proliferation and estradiol production were not affected by DIBP treatments in combination with FSH or FSH+IGF1. DIBP treatment in combination with FSH+IGF1 decreased mRNA levels of METTL14 (P<0.01), an RNA N6 methyladenosine (m6A) methylation methyltransferase, TRDMT1 (P<0.05), an RNA 5-methylcytosine (m5C) methylation methyltransferase, and ASC (P<0.05), an NLRP3 inflammasome player. These results indicate that DIBP at environmentally relevant doses regulates gene expression of epigenetic and inflammatory pathways but does not alter GC proliferation or steroidogenesis. The second project aimed to characterize the phthalate concentrations on a livestock farm. Phthalate concentrations were measured in alfalfa hay, corn silage, and pasture. Lactating multiparous Angus cows were fed with alfalfa hay or corn silage for 49 hours. Cows were maintained on pasture prior to exposure to other types of feed. Urine and blood samples were collected before and during feedstuff exposure to examine phthalate metabolite levels. Silage presented higher concentrations of Di-ethyl phthalate (DEP), Benzylbutyl phthalate (BBP), and Di-isononyl phthalate (DINP) than pasture and hay (P<0.05), and hay presented higher concentrations of Di-n-octyl phthalate (DOP) and DINP than pasture (P<0.05). During the trial, hay-fed group had higher urine concentrations of Mono-isobutyl phthalate (MIBP), Mono-n-butyl phthalate (MBP), Mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), Mono-2-ethylhexyl terephthalate (MEHtP) compared with silage-fed group (P<0.05), and silage fed group had higher urine concentrations of Mono-n-pentyl phthalate (MPP), Mono-cyclohexyl phthalate (MCHP), Mono-2-ethylhexyl phthalate (MEHP), Mono-isononyl phthalate (MINP) (P<0.05). Urine concentrations of MCHP and Mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) were higher in silage-fed group at the end of the trial compared with prior to the start of the trial (P<0.05). Urine concentrations of MIBP and MEOHP were higher in hay fed group at the end of the trial compared with prior to the start of the trial (P<0.05). No differences in plasma phthalate metabolite concentrations were found between hay-fed group and silage-fed group after 49 hours of feeding. However, because Mono-benzyl phthalate (MBzP) had the highest plasma concentrations, it was selected to determine its effects on in vitro cultured bovine GC function. Bovine GC were treated with MBzP at concentrations found in the plasma in the animal trial combined with FSH+IGF1. Bovine GC steroidogenesis or proliferation were not affected by MBzP treatments. The findings demonstrated that silage and, to a lesser extent, hay are possible sources of phthalate exposure for cattle and that feeding them silage or hay resulted in greater amounts of several phthalates. Taken together, these studies reinforce the importance of monitoring environmental contaminants in livestock production and assessing the effects of phthalates at environmentally relevant doses on human and animal reproduction.